The E3 Ubiquitin Ligase SIAH1 Targets MyD88 for Proteasomal Degradation During Dengue Virus Infection

Abstract

The dengue virus presents a serious threat to human health globally and can cause severe, even life-threatening, illness. Dengue virus (DENV) is endemic on all continents except Antarctica, and it is estimated that more than 100 million people are infected each year. Herein, we further mine the data from a previously described screen for microRNAs (miRNAs) that block flavivirus replication. We use miR-424, a member of the miR-15/16 family, as a tool to further dissect the role of host cell proteins during DENV infection. We observed that miR-424 suppresses expression of the E3 ubiquitin ligase SIAH1, which is normally induced during dengue virus 2 (DENV2) infection through activation of the unfolded protein response (UPR). Specific siRNA-mediated knockdown of SIAH1 also results in inhibition of DENV replication, demonstrating that this target is at least partly responsible for the antiviral activity of miR-424. We further show that SIAH1 binds to and ubiquitinates the innate immune adaptor protein MyD88 and that the antiviral effect of SIAH1 knockdown is reduced in cells in which MyD88 has been deleted by CRISPR/Cas9 gene editing. Additionally, MyD88-dependent signaling, triggered either by DENV2 infection or the Toll-like receptor 7 (TLR7) ligand imiquimod, is increased in cells in which SIAH1 has been knocked down by miR-424 or a SIAH1-specific siRNA. These observations suggest an additional pathway by which DENV2 harnesses aspects of the UPR to dampen the host innate immune response and promote viral replication.

Keywords: dengue; flavivirus; microRNA; ubiquitin; unfolded protein response.

FIGURE 1

MicroRNAs (miRNAs) of the miR-15/16 family with identical seed sequences inhibit dengue virus 2 (DENV2). (A) Comparison of the seed sequences of the members of the miR-15/16 family of miRNAs. Identical seed sequences (nucleotides 2–7) are highlighted in blue; the seed sequences of miR-103 and miR-107 (in green) are similar but offset by one nucleotide. (B) HeLa cells were transfected with the indicated miR-15/16 family member duplex RNA mimics, a validated non-targeting miRNA mimic (miR-Ct) as a negative control, or a DENV2 genome siRNA (DENV2 siR) and infected with DENV2 (MOI = 3 ffu/cell) 2 days post-transfection. Supernatants were collected 3 days p.i. and titered on Vero cells.

FIGURE 2

Expression of a miR-424 mimic inhibits RNA virus infection. (A) HeLa cells were transfected with a duplex RNA mimic of miR-424, the broadly antiviral miR-199a-3p, an siRNA against the DENV2 genome (DENV2 siRNA), or a negative control miRNA mimic (miR-Ct). Two days post-transfection, cells were infected with DENV2 (MOI = 10 ffu/cell) or WNV (MOI = 3 ffu/cell). Two (WNV) or 3 (DENV2) days p.i., cells were fixed and incubated with anti-flavivirus E antibody, and infected cells visualized with an Alexa Fluor–conjugated anti-mouse immunoglobulin antibody (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Supernatants were collected from the above infected cells at 2 h, 1 day, and 2 days p.i., and infectious viral titers [displayed as focus-forming unit (ffu)/ml] determined by focus-forming assay on Vero cells. (C) HeLa cells transfected with the miR-424 mimic or negative control miRNA mimic were infected with CHKV, vesicular stomatitis virus (VSV), herpes simplex virus (HSV), or vaccinia virus (VacV) (MOI = 0.5 pfu/cell). Supernatants were collected 3 days p.i., and titers [displayed as plaque-forming unit (pfu)/ml] determined by plaque-forming assay on Vero cells. Results are representative of at least three independent experiments. (* p-value < 0.05, ** p-value < 0.01, **** p-value < 0.001).

FIGURE 3

miR-424 targets the 3′ UTR of SIAH1, SMURF1, and SMURF2, inhibiting transcript expression in uninfected and DENV2 infected cells. (A) Dual luciferase reporter plasmids were constructed containing the 3′ UTRs of SIAH1, SMURF1, or SMURF2. The 3′ UTR of SMURF1 contains two putative miR-424 binding sites, prompting the construction of two plasmids containing one binding site each. HEK293 cells were transfected with the reporter plasmid and the miR-424 mimic or control small RNA. Cells were lysed 48 h post-transfection, and luciferase expression measured by luminometer. Activity is represented as relative luciferase units (RLUs), calculated as test normalized to control. (B) HeLa cells were transfected with siRNA against SMURF1 or SMURF2, the miR-424 mimic, or a control mimic, and 48 h post-transfection total RNA was collected in Trizol. Relative expression of the SMURF1 and SMURF2 mRNAs was determined by qPCR and normalized to β-actin. (C) HeLa cells were transfected with the SIAH1 siRNA, the miR-424 mimic, or control mimic. At 48 h post-transfection, they were treated with thapsigargin (1 μM) for 6 h. Total RNA in Trizol was collected and relative mRNA levels of SIAH1 determined as described above. (D) HeLa cells were transfected as indicated and infected with DENV2 (MOI = 5 ffu/cell). Total RNA was collected in Trizol at the indicated time points and relative mRNA levels determined as described above. (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005, **** p-value < 0.001).

FIGURE 4

Inhibition of SIAH1 expression recapitulates the antiviral effect of miR-424 during DENV2 infection. HeLa cells (A) or Huh7 cells (B) were transfected as indicated and infected with DENV2 (MOI = 5 ffu/cell) 48 h post-transfection. Supernatants from infected cells were collected 48 h p.i. and titered on Vero cells. (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005).

FIGURE 5

SIAH1 knockdown inhibits proteasome-dependent degradation of MyD88. (A) HeLa cells were transfected with as indicated and infected with DENV2 (MOI = 5 ffu/cell) 48 h post-transfection. At 42 h p.i., a cohort of siRNA control transfected wells were treated with MG132 to inhibit the proteasome. Cell lysates were collected 48 h p.i. and analyzed by western blot for MyD88 expression. (B) Wild-type (WT) and MyD88-deficient HeLa cells were transfected as indicated, infected with DENV2 (MOI = 5 ffu/ml) 48 h post-transfection. Supernatants were collected 48 h p.i. and titered on Vero cells. Fold reduction in viral titer represents the ratio of ffu/ml from control cells to transfected cells. MyD88 knockout was confirmed by western blot (inset). (* p-value < 0.05, *** p-value < 0.005).

FIGURE 6

SIAH1 binds and ubiquitinates MyD88. (A) HeLa cells were treated with MG132 for 6 h and then transfected with plasmids expressing SIAH1-FLAG and MyD88-myc in the presence of MG132. Twenty-four hours post-transfection, total cell lysates were collected, immunoprecipitated with α-FLAG or an isotype control antibody, and analyzed by western blot (TCL, total cell lysate). (B) HeLa cells were transfected with or without SIAH1 siRNA as well as with plasmids expressing MyD88-FLAG and Ub-myc. Twenty-four hours post-transfection, cells were treated with thapsigargin to induce SIAH1 expression, and 2 days post-transfection with MG132 to inhibit the proteasome. Total cell lysates were immunoprecipitated with α-myc and analyzed by western blot.

FIGURE 7

Inhibition of SIAH1 increases expression of NF-κB- and interferon-induced genes. (A–D) HeLa cells were transfected with the indicated siRNA or miRNA mimic and infected with DENV2 (MOI = 5 ffu/ml) 48 h post-transfection. Total RNA was collected in Trizol 48 h p.i., and relative transcripts levels determined by qPCR normalized to β-actin and uninfected control mRNA levels. (E) 48 h after transfection, cells were treated with imiquimod (10 μg/ml) for 24 h. Total RNA was collected and relative mRNA levels determined as described above. (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005, **** p-value < 0.001).